WO2023094429A1 - Régions promotrices artificielles de levure - Google Patents

Régions promotrices artificielles de levure Download PDF

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WO2023094429A1
WO2023094429A1 PCT/EP2022/082939 EP2022082939W WO2023094429A1 WO 2023094429 A1 WO2023094429 A1 WO 2023094429A1 EP 2022082939 W EP2022082939 W EP 2022082939W WO 2023094429 A1 WO2023094429 A1 WO 2023094429A1
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protein
interest
yeast
expression
promoter region
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Heiko DIETZ
Julia Bauer
Melissa KRACHT
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Kaesler Nutrition Gmbh
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/102—Plasmid DNA for yeast
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/15—Vector systems having a special element relevant for transcription chimeric enhancer/promoter combination

Definitions

  • the invention relates to methods for optimizing expression of a protein of interest in a yeast host cell.
  • Yeasts such as Saccharomyces cerevisiae and Schizosaccharomyces pom.be are renowned as production facilities for a protein of interest. Deletion mutants of every open reading frame (Scherens and Goffeau, 2004. Genome Biol 5: 229; Kim et al., 2010. Nat Biotechnol 28: 617-623) have resulted in a thorough understanding of the biological roles of many of the genes.
  • the Saccharomyces Genome Database (SGD; available at yeastgenome.org/) provides information about each and every yeast gene, including the effects of over- and under-expression or deletion of a gene.
  • yeast species that are adapted to the process conditions and can tolerate the mechanical forces in a bioreactor, inhibitory substances and fermentation products.
  • divers yeast products have obtained GRAS (generally recognized as safe) status by the FDA, while the European Food Safety Authority (EFSA) has provided a list of organisms, termed Qualified Presumption of Safety (QPS), which includes several yeast strains.
  • GRAS generally recognized as safe
  • EFSA European Food Safety Authority
  • yeasts The knowledge of intracellular processes such as metabolism, secretion, transport, signaling and other pathways, and the availability of a large tool set for genetic engineering, have made yeasts the workhorse for a wide variety of applications.
  • heterologous protein requires control over gene expression to optimize product formation.
  • Transcriptional control for example the strength of a promoter and possible feedback mechanisms, are critical points for yeast engineering.
  • This new generation of yeast promoter sequences preferably drive expression of a protein at different levels, allowing expression of a gene at a requested level.
  • the invention described herein is based on numerous artificial yeast promoter regions, comprising different promoter elements such as enhancer elements and TATA boxes at varying positions, that were tested in different yeast strains. These tests resulted in the identification of a minimal number of promoter elements that provide tailored expression levels of a protein in the yeast strains tested.
  • the invention provides an artificial yeast promoter region, comprising a TATA box at position -90 ⁇ 15 nucleotides (nt), and/or a TATA box at position -160 ⁇ 15 nt, an enhancer element at position -350 ⁇ 25 nucleotides, and a second enhancer element at position -600 ⁇ 50 nucleotides, wherein said enhancer element at position -350 ⁇ 25 nucleotides is selected from a Asparagine -rich Zinc-Finger 1 (AZFI) binding element and a Multicopy suppressor of SNF1 mutation (MSN4) binding element, wherein said enhancer element at position -600 ⁇ 50 nucleotides is selected from a GlyColysis Regulation 1 (GCR1) binding element, a GCR2 binding element, and a PseudoHyphal Determinant 1 (PHD1) binding element, wherein all positions are relative to the start codon ATG, and wherein the sequences in between the indicated TATA boxes and enhancer elements
  • the enhancer elements are not positioned at their natural distance to the start codon.
  • the enhancer element at position -350 ⁇ 25 nucleotides is preferably 5’-AAMRGMA or 5’-RVCCCCYR.
  • the second enhancer element at position -600 ⁇ 50 nucleotides is preferably selected from 5’-WGGAWGMY, 5’-WGGAAGNM, and 5’-VMTGCRKV.
  • TATA box is present at position -90 ⁇ 15 nucleotides (nt), at position -160 ⁇ 15 nt, or at both positions - 90 ⁇ 15 nucleotides (nt) and -160 ⁇ 15 nt.
  • Said artificial yeast promoter region is able to drive expression of a downstream protein in at least one of Kluyverontyces marxianus, Kluyveromyces lactis, Komagataella pastoris, Komagataella phaffii, Ogataea angusta, Yarrowia lipolytica, Schizosaccharomyces pom.be, Rhodotorula mucilaginosa, Candida famata and Saccharomyces cerevisiae, preferably at least in one of K phaffii, Y. lipolytica and S. cerevisiae.
  • the nucleotide sequences of the promoter region are preferably identical to any one of SEQ ID NO:s 1-33, over a length of at least 600 nucleotides.
  • the invention further provides an expression construct comprising the artificial yeast promoter region of the invention, for expression of a protein of interest in a yeast.
  • Said expression construct preferably comprises a nucleotide sequence encoding a protein of interest under control of the artificial yeast promoter region.
  • Said nucleotide sequence encoding the protein of interest preferably is codon optimized for expression in a yeast host cell.
  • the invention further provides a yeast host cell, comprising the artificial yeast promoter region of the invention, or the expression construct according the invention.
  • the invention further provides a method of producing a protein of interest in a yeast host cell, comprising providing an expression construct according to the invention, transforming a yeast cell with the expression construct, expressing the protein of interest; and, optionally, at least partly purifying the protein of interest.
  • the invention further provides method of producing a protein of interest in a yeast host cell, comprising providing the yeast host cell comprising the expression construct according to the invention, expressing the protein of interest; and, optionally, at least partly purifying the protein of interest.
  • the protein of interest is preferably tagged.
  • the protein of interest may be co-expressed in the yeast cell with one or more of a protein disulfide isomerase, a flavin-linked sulfhydryl oxidase, and an oxidoreductase.
  • the protein of interest may be part of a metabolic pathway, the method further comprising modulating the expression levels of one or more other enzymes in the metabolic pathway.
  • Said metabolic pathway may be selected from the production of a biofuel, the breakdown of a carbohydrate, the production of a biopolyester, the production of a tocochromanol, and the production of an alkaloid.
  • Fig. 1 Cultivation of K. phaffii in media containing three different carbon sources to express YFP as a reporter under control of different promoters with a high strength.
  • Fig. 2 Cultivation of K. phaffii in medium containing two different carbon sources to express YFP as a reporter under control of various promoters with a medium strength.
  • Fig. 3 Cultivation of Y. lipolytica to express GFP as a reporter under control of the different promoters as indicated with a high strength (A), or with a similar fluorescence (B), when compared to a reference promoter.
  • Fig. 4 Cultivation of S. cerevisiae to express YFP as a reporter under control of the different promoters with a high strength.
  • Fig. 5 Cultivation of S. cerevisiae to express YFP as a reporter under control of the different promoters with a medium strength.
  • Fig. 6 Cultivation of S. cerevisiae for production of geranylgeraniol under control of the promoter EPK14 compared to the reference promoter.
  • Fig. 7 Cultivation of K. phaffii in media containing three different carbon sources for expression of phytase AppA from E. coli under control of the promoters EPK2 and EPK3 compared to the reference promoter.
  • Fig. 8 Normalized specific activity of phytase with PDI co-expression compared to the AppA production strain, which did not express PDI.
  • substantially(ly) refers to the general character or function which is specified. When referring to a quantifiable feature, these term is in particular used to indicate that it is for at least 75 %, more in particular at least 90 %, even more in particular at least 95 % of the indicated feature.
  • a “substantially pure” compound or protein refers to a purity of at least 95%, more preferably at least 96 %, at least 97 %, at least 98 %, at least 99 % or at least 100 % pure compound or protein, as determined by standard analytical techniques known in the art.
  • yeast refers to a eukaryotic, unicellular microorganism that is classified as a member of the kingdom fungus.
  • a preferred yeast is a yeast of the Saccharomyces sensu stricto complex (Hittinger, 2013. Trends Genet 29: 309-317; Naseeb et al., 2017.
  • Int J Syst Evol Microbiol 67: 2046- 2052) such as the species Saccharomyces cerevisiae, a methylotrophic yeast such as Komagataella pastoris, Komagataella phaffii (both together formerly known as Pichia pastoris) and Ogataea angusta (formerly known as Hansenula polymorpha), a fission yeast such as Schizosaccharomyces pom.be, a Kluyveromyces species such as K. lactis and K. marxianus, a Yarrowia species such as Y. lipolytica, and a Arxula species such as Arxula adeninivorans.
  • Saccharomyces cerevisiae a methylotrophic yeast such as Komagataella pastoris, Komagataella phaffii (both together formerly known as Pichia pastoris) and Ogataea angusta
  • a fission yeast
  • gene refers to a nucleic acid molecule comprising a protein-coding or RNA-coding sequence such as miRNA or siRNA, in an expressible form, operably linked to a regulatory sequence, including a promoter and optionally other regulatory sequences that are required to control expression of the coding sequence.
  • heterologous gene refers to any gene or coding sequence of a gene that does not naturally occur in the species wherein it is expressed.
  • transformation refers to the introduction of a nucleotide sequence such as a plasmid into a cell.
  • the term preferably refers to stable integration of a nucleotide sequence such as a plasmid into the genome of a cell.
  • encoding refers to the information to guide translation of a nucleotide sequence of a nucleic acid molecule into a specified protein.
  • the information by which a protein is encoded is specified by codons, i.e. a trinucleotide sequence of DNA or RNA that corresponds to a specific amino acid.
  • a nucleic acid molecule encoding a protein may comprise a non-translated sequence, e.g., an intron, interspersed with translated regions of the nucleic acid or may lack such an intervening non-translated sequence, e.g., as in complementary DNA (cDNA).
  • cDNA complementary DNA
  • mRNA messenger RNA
  • miRNA microRNA
  • operably linked refers to the association of two or more nucleic acid fragments on a single nucleic acid molecule so that the function of one is affected by the other.
  • a promoter region is operably linked to a coding sequence when the promoter region is capable of affecting the expression of said coding sequence.
  • the coding sequence is under transcriptional control of the promoter region.
  • express refers to the process of transcription or/or translation of a gene.
  • promoter region refers to a nucleotide sequence upstream or surrounding a transcription start site that controls binding of an RNA polymerase.
  • a coding sequence or functional RNA is located downstream of a promoter.
  • TATA box refers to an element in the promoter region that functions as initiating site for a transcription complex comprising TATA-binding protein.
  • a TATA box consensus sequence comprises the nucleotide sequence 5’-TATA(A/T)A(A/T)(A/G), and thus includes the sequences 5'- TATAAAAA, 5'-TATAAAAG, 5'-TATAAATA, 5'-TATAAATG, 5'-TATATAAA, 5'- TATATAAG, 5'-TATATATA and 5'-TATATATG.
  • small alterations from this consensus sequence such as 1 or 2 alterations, may be tolerated.
  • regulatory element refers to a nucleotide sequence located upstream, interspersed with, or downstream of a promoter that influences transcription, RNA processing or stability, or translation of the associated coding sequence.
  • a regulatory element may include an enhancer element, translational leader sequence (5’ untranslated regions (UTR), intron, trailer sequence (3’ UTR), and a polyadenylation sequence.
  • enhancer element refers to a specific nucleotide sequence that can stimulate promoter activity. In general, an enhancer functions independent of its exact position relative to a promoter.
  • alteration refers to a change in a nucleic acid sequence of a gene compared to the nucleic acid sequence of a non- altered gene. Said change preferably is a change that affects the functionality of the gene. Encompassed in the term alteration is deletion of one or more, including all, nucleotides from the gene, substitution of one or more nucleotides in the gene, and insertion of one or more nucleotides into the gene, or a combination thereof.
  • substitution refers at a nucleic acid level to a change of one or more nucleotides into one or more other nucleotides, whilst the total number of nucleotides remain the same.
  • substitution at the amino acid level is a change of one or more amino acid residues into one or more other amino acid residues, whilst the total number of amino acid residues remains the same.
  • protein refers to a chain of amino acids arranged in a specific order determined by the coding sequence in a nucleic acid molecule encoding the protein.
  • sequence identity refers to the percentage of identical residues determined by comparing two optimally aligned sequences over a comparison window. Said two sequences are preferably compared over the full length of the shortest of two sequences. The percentage is calculated by determining the number of positions at which an identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity.
  • purifying refers to at least partly separating of a compound or protein of interest from its environment. Purifying encompasses separating a fluid comprising said compound or protein of interest from solids such as cells and debris to obtain a fluid comprising the compound or protein of interest and optionally one or more other substances. Purifying further encompasses removal of impurities and other substances until the compound or protein of interest is substantially pure.
  • This invention provides an artificial yeast promoter region, comprising one or two TATA boxes and two enhancer elements.
  • a first TATA box is present at position -90 ⁇ 15 nucleotides (nt) relative to the translation start codon ATG, while a second TATA box is present at position -160 ⁇ 15 nt.
  • Said artificial promoter region is further characterized by the presence of two enhancer elements, a first element at position -350 ⁇ 25 nucleotides, and a second enhancer element at position -600 ⁇ 50 nucleotides.
  • nucleotide sequences in between the indicated TATA boxes and enhancer elements were shuffled sequences from existing yeast promoter regions. Care was taken when designing these promoter regions to eliminate possible transcriptional repressor elements such as ACR1 (5’-ATGACGTCA; Vincent and Struhl, 1992. Mol Cell Biol 12: 5394-5405), Roxl QiNTVGWN,- Pevny and Lovell-Badge, 1997. Curr Opin Genet Dev 7: 338-344), and Rgt 1 (5'-CGGANNA; Kim, 2009. Biochimie 91: 300-303).
  • A represents adenine
  • G represents guanine
  • C represents cytosine
  • T represents thymine
  • Y represents a pyrimidine (C or T)
  • R represents a purine (A or G)
  • W represents weak (A or T)
  • S represents strong (G or C)
  • K represents keto (T or G)
  • M represents amino (C or A)
  • D represents A, G, T (not C)
  • V represents A, C, G (not T)
  • H represents A, C, T (not G)
  • B represents C, G, T (not A)
  • X or N represents any base.
  • a gap is denoted by a
  • Said first and second enhancer elements may be any enhancer element, provided that the first enhancer element differs from the second enhancer element, and that the first and second enhancer elements bind different transcription activating proteins.
  • said first and second enhancer element preferably bind a transcription activating protein in substantially all yeasts, preferably at least in one or more of Saccharomyces cerevisiae, Komagataella pastoris, Komagataella phaffii, Ogataea angusta, Schizosaccharomyces pom.be, Kluyveromyces lactis, Kluyveromyces marxianus, Yarrowia lipolytica, Rhodotorula mucilaginosa and Candida famata.
  • Said first and second enhancer elements are not positioned at their natural position relative to the start codon ATG.
  • MSN4 encodes a Cys2His2 zinc finger protein that binds to a stress- responsive element termed STRE in the promoter region of several genes, having the consensus sequence 5’-RVCCCCYR, normally resides at around position.
  • An artificial yeast promoter region according to the invention may be provided by fixing two TATA boxes at positions -90 ⁇ 15 nucleotides (nt) and/or - 160 ⁇ 15 nt, relative to the translation start codon ATG, a first enhancer element at position -350 ⁇ 25 nucleotides, and a second enhancer element at position -600 ⁇ 50 nucleotides.
  • the sequences in between these elements may be obtained from common yeast promoter regions, but are preferably shuffled or scrambled so that they sufficiently differ from existing promoter sequences.
  • a total length of a promoter region according to the invention is between 600 and 1200 nt, such as between 700 and 1000 nt, such as around 800 nt.
  • Said first and second enhancer element may provide a binding site for any known yeast transcription factor such as, for example, Sterile 12 (Stel2), Heme Activator Protein 1 (Hapl), Asparagine -rich Zinc-Finger 1 (AZFI; YOR113W), Multicopy suppressor of SNF1 mutation (MSN4), GlyColysis Regulation 1 (GCR1), GCR2 and PseudoHyphal Determinant 1 (PHD1; YKE043W).
  • Stel2 Sterile 12
  • Hapl Heme Activator Protein 1
  • AZFI Asparagine -rich Zinc-Finger 1
  • MSN4 Multicopy suppressor of SNF1 mutation
  • GCR1 GlyColysis Regulation 1
  • PseudoHyphal Determinant 1 PseudoHyphal Determinant 1
  • a preferred artificial yeast promoter region according to the invention comprises a first enhancer element at position -350 ⁇ 25 nucleotides selected from an Asparagine-rich Zinc-Finger 1 (AZFI; YOR113W) binding element and a Multicopy suppressor of SNF1 mutation (MSN4) binding element.
  • AZFI Asparagine-rich Zinc-Finger 1
  • MSN4 Multicopy suppressor of SNF1 mutation
  • AZFI is a Cys2His2 zinc finger transcription factor that activates transcription of genes involved in carbon metabolism, energy production on glucose, cell wall organization and biogenesis on glycerol-lactate.
  • AZFI binds a consensus sequence 5’-AAMRGMA, preferably 5’-AAAAGAA.
  • MSN4 (YKL062W) is also a zinc finger protein that activates genes in response to several stresses, including heat shock, osmotic shock, oxidative stress, low pH, glucose starvation, sorbic acid and high ethanol concentrations. MSN4 binds to a consensus sequence 5’-CCCCT, preferably 5’-RVCCCCYR.
  • a preferred artificial yeast promoter region according to the invention comprises a second enhancer element at position -600 ⁇ 50 nucleotides is selected from a GCR1 binding element, a GCR2 binding element, and a PHD1 binding element.
  • GlyColysis Regulation 1 (GCR1; YPL075W) is a transcriptional activator that drives expression of glycolytic and ribosomal genes. GCR1 binds to a core 5'- GGAAG sequence, termed CT box, preferably 5’-WGGAWGMY).
  • GCR2 (YNL199C) also is a transcriptional activator that drives expression of glycolytic and ribosomal genes. GCR2 interacts and functions with the DNA-binding protein GCR1. GCR2 binds to a core 5’-WGGAAGNM sequence.
  • PseudoHyphal Determinant 1 (PHD1; YKL043W) is a transcriptional activator that enhances pseudohyphal growth. PHD1 binds to a core 5’- VMTGCRKV sequence.
  • a preferred artificial yeast promoter region is able to drive expression of a downstream protein in several yeasts.
  • Said yeasts preferably include at least one of Kluyverontyces marxianus, Kluyveromyces lactis, Komagataella pastoris, Komagataella phaffii, Ogataea angusta, Yarrowia lipolytica, Schizosaccharomyces pom.be, Rhodotorula mucilaginosa and Candida famata. and Saccharomyces cerevisiae, preferably at least in one of K. phaffii, Y. lipolytica and S. cerevisiae, more preferably at least two of K. phaffii, Y. lipolytica and S. cerevisiae.
  • a preferred artificial yeast promoter region comprises a nucleotide sequence that is at least 60 % identical to one or more of SEQ ID NO:s 1-33, preferably at least 80 % identical to one or more of SEQ ID NO:s 1-33, over a length of at least 600 nucleotides.
  • Said preferred artificial yeast promoter region comprises a nucleotide sequence that is at least 85 % identical, at least 90 % identical, at least 91 % identical, at least 92 % identical, at least 93 % identical, at least 94 % identical, at least 95 % identical, at least 96 % identical, at least 97 % identical, at least 98 % identical, at least 99 % identical, at least 100 % identical to one or more of SEQ ID NO:s 1-33 over a continuous stretch of at least 600 nucleotides, more preferred at least 650 nucleotides, more preferred at least 700 nucleotides, more preferred at least 750 nucleotides, more preferred over their full length to one or more of SEQ ID NO:s 1-33.
  • the invention further provides a method of producing an artificial yeast promoter region, comprising a TATA box at position -90 ⁇ 15 nucleotides (nt) and/or a TATA box at position -160 ⁇ 15 nt, an enhancer element at position -350 ⁇ 25 nucleotides, and a second enhancer element at position -600 ⁇ 50 nucleotides, wherein all positions are relative to the start codon ATG, wherein the enhancer elements are not positioned at their natural distance to the start codon, and wherein the sequences in between the indicated TATA boxes and enhancer elements lack known repressor elements.
  • Said first enhancer element at position -350 ⁇ 25 nucleotides preferably is selected from AZFI (5’-AAMRGMA) and MSN4 (5’-RVCCCCYR).
  • Said second enhancer element at position -600 ⁇ 50 nucleotides preferably is selected from a GCR1 binding element, preferably 5’-WGGAWGMY, a GCR2 binding element, preferably 5’-WGGAAGNM, and a PHD1 binding element, preferably 5’-VMTGCRKV.
  • sequences in between the indicated TATA boxes and enhancer elements may be selected from existing yeast promoter regions and preferably comprise shuffled sequences from existing yeast promoter regions.
  • Known sequences that may bind to transcriptional repressor elements such as ACR1 (5’-ATGACGTCA; Vincent and Struhl, 1992. Mol Cell Biol 12: 5394-5405), Roxl (WTTGWW; Pevny and Lovell-Badge, 1997. Curr Opin Genet Dev 7: 338-344), and Rgt 1 (5'- CGGANNA; Kim, 2009. Biochimie 91: 300-303), preferably are avoided.
  • an artificial yeast promoter region preferably is adjusted to mimic the GC-content of a yeast host organism.
  • an artificial yeast promoter region may comprise a GC content of between 30-50 %.
  • a GC content of 49 % for optimal expression in S. cerevisiae (GC content of 38.3 %), an artificial yeast promoter region may comprise a GC content of between 40-60 %.
  • the invention further provides an expression construct comprising an artificial yeast promoter region of the invention, for expression of a protein of interest in a yeast.
  • the expression construct preferably comprises a nucleotide sequence encoding a protein of interest under control of the artificial yeast promoter region.
  • Said expression construct is either a construct that integrates into the yeast host genome, preferably at a pre-selected position in the yeast host genome, or an episomal expression vector.
  • Expression constructs typically contain a yeast promoter and terminator sequences and a yeast selectable marker cassette, as is known to a person skilled in the art.
  • Most yeast vectors can be propagated and amplified in E. coli to facilitate cloning.
  • these constructs also contain an E. coli origin of replication and a selectable marker such as, for example, a betalactamase (Bia) gene for resistance to ampicillin.
  • an yeast expression construct may include a secretion leader amino acid sequence that efficiently directs a protein of interest outside of the yeast host cell into the growth medium.
  • Said protein of interest is preferably selected from a pharmacologically active protein, an antibody or antibody fragment, a therapeutic protein, a peptide such as peptide hormone or an antimicrobial peptide, an enzyme such as a cellulase, a protease, a protease inhibitor, an aminopeptidase, an amylase, a carbohydrase, a carboxypeptidase, a catalase, a chitinase, a cutinase, a deoxyribonuclease, an esterase, an alpha-galactosidase, a beta- galactosidase, a glucoamylase, an alphaglucosidase, a beta-glucosidase, an invertase, a laccase, a lipase, a mannanase, a mutanase, an oxidase, a pectinolytic
  • a nucleotide sequence encoding a protein of interest is preferably codon optimized or codon harmonized for expression in a yeast.
  • the term “codon optimized”, as is used herein, refers to the selection of codons in a nucleotide sequence encoding a protein of interest for optimal expression of the protein of interest in a yeast host cell.
  • a single amino acid may be encoded by more than one codons. For example, arginine and leucine are each encoded by a total of 6 codons. Codon optimization, i.e. the selection of a preferred codon for a specific yeast cell at every amino acid residue, plays a critical role, especially when proteins are expressed in a heterologous system.
  • codon optimization may play a role in achieving high gene expression levels, other factors such as secondary structure of the messenger RNA also need to be considered. Codon optimization is offered by commercial institutions, such as ThermoFisher Scientific, called Invitrogen GeneArt Gene Synthesis, GenScript, called GenSmartTM Codon Optimization, or GENEWIZ, called GENEWIZ’s codon optimization tool.
  • Codon harmonized refers to the alignment of codon usage frequencies with those of the expression yeast, particularly within putative inter-domain segments where slower rates of translation may play a role in protein folding. Codon harmonization may be accomplished by algorithms such as provided by Angov et al., 2011 (Angov et al., 2011. In: Evans and Xu (eds) Heterologous Gene Expression in E. coli. Methods in Molecular Biology (Methods and Protocols), vol 705. Humana Press; Claassens et al., 2017. PLoS One 12: e0184355.
  • gene products expressed from heterologous genes maybe more prone to degradation caused by proteolytic activity than proteins that are naturally expressed in a yeast cell. Such proteins do not naturally occur in the environment wherein they are secreted and may thus be more prone to degradation. Care has to be taken not to include proteolytic cleavage sites in the protein of interest for a proteolytic enzyme from the host yeast cell.
  • one or more conserved amino acid alterations in which an aliphatic amino acid residue is replaced for another aliphatic amino acid residue, a hydroxyl or sulfur/selenium-containing amino acid residue is replaced for another hydroxyl or sulfur/selenium-containing amino acid residue, an aromatic amino acid residue is replaced for another aromatic amino acid residue, a basic amino acid residue is replaced for another basic amino acid residue, and an acidic amino acid residue or amide thereof is replaced for another acidic amino acid residue or amide thereof, may be engineered in the protein of interest to efficiently stop degradation.
  • Said conserved amino acid replacements can be made for aliphatic amino acid residues glycine, alanine, valine, leucine, and isoleucine; hydroxyl or sulfur/selenium-containing amino acid residues serine, cysteine, selenocysteine, threonine and methionine, aromatic amino acid residues phenylalanine, tyrosine, and tryptophan, basic amino acid residues histidine, lysine, and arginine; and acidic amino acid residues and their amides aspartate, glutamate, asparagine, and glutamine.
  • the invention further provides a yeast host cell, comprising an artificial yeast promoter region or an expression construct according to the invention.
  • Said yeast host cell preferably is selected from Candida hispaniensis, Kluyveromyces marxianus, Kluyveromyces lactis, Komagataella phaffii, Ogataea angusta, Yarrowia lipolytica, and Saccharomyces cerevisiae, more preferably K phaffii, Y. lipolytica or S. cerevisiae.
  • the invention further provides a method of producing a protein of interest in a yeast host cell, comprising providing an expression construct according to invention, wherein the expression of a protein of interest is controlled by an artificial yeast promoter region according to the invention, transforming a yeast cell with the expression construct; expressing the protein of interest; and, optionally, at least partly purifying the protein of interest.
  • Said transformation preferably involves the integration of the expression construct in the genome of the yeast host cell.
  • This integration may be performed randomly, or at a chosen locus of the genome of the yeast host cell by homologous recombination, for example through the use of Cre/Zox or a CRISPR-Cas recombination system.
  • the robust and precise integration of an expression construct at a specific locus of the yeast genome allows stable, high expression levels of a protein of interest in a yeast host cell. Said high expression levels in general are reproducible for different proteins of interest.
  • the invention further provides a method of producing a protein of interest in a yeast host cell, comprising an expression construct wherein the expression of a protein of interest is controlled by an artificial yeast promoter region according to the invention, expressing the protein of interest; and, optionally at least partly purifying the protein of interest.
  • a yeast host cell expressing the protein of interest under control of an artificial yeast promoter region may be selected that has correctly integrated the expression that expresses the protein of interest.
  • a selected yeast host cell may be grown, for example, in fed-batch or fermenter cultures, to very high cell densities reaching up to 150 g cell dry weight per litre.
  • Purification of a protein of interest may comprise a series of processes intended to isolate one or more proteins from a complex mixture, usually cells, tissues, and/or growth medium.
  • Various purification strategies can be followed.
  • proteins can be separated based on size, for example in a method called size exclusion chromatography.
  • proteins can be purified based on charge, e.g. through ion exchange chromatography or free-flow- electrophoresis, or based on hydrophobicity (hydrophobic interaction chromatography). It is also possible to separate proteins based on molecular conformation, for example by affinity chromatography.
  • Said purification may involve the use of a specific tag, for example at the N-terminus and/or C-terminus of the protein.
  • the proteins may be concentrated. This can for example be carried out with lyophilization or ultrafiltration.
  • a protein of interest may be tagged in order to facilitate purification of the protein.
  • a tag refers to the addition of a peptide to the amino (N) or carboxy (C) terminus of a protein. The addition of a tag allows to isolate or immobilize a protein.
  • Commonly used tags include a poly-histidine tag such as a 6x(His) tag, a myc tag, a glutathione-S-transferase tag, a HiBiT tag (Promega, Madison, Wisconsin), a FLAG tag, a HA tag, or multimeric tags such as a triple FLAG tag If required, a protein of interest may be co-expressed in a yeast host cell together with one or more proteins that may increase correct folding of a recombinantly expressed protein of interest in a yeast host cell.
  • one or more proteins such as a protein disulfide isomerase, for example protein disulfide isomerase PDI1 (YCL043C), ER protein Unnecessary for Growth (EUG1, YDR518W), or Multicopy suppressor of PDI1 deletion (MPD1; YOR288C); a flavin- linked sulfhydryl oxidase, for example Essential for Respiration and Viability (ERV2; YPR037C); and /or thiol oxidase such as ER Oxidation or Endoplasmic Reticulum Oxidoreductin (ERO1; YML130C), may be overexpression in a yeast host cell to assist in correct folding of a protein of interest.
  • a protein disulfide isomerase for example protein disulfide isomerase PDI1 (YCL043C), ER protein Unnecessary for Growth (EUG1, YDR518W), or Multicopy suppressor
  • Genes encoding one or more of the indicated Saccharomyces genes, or a related gene from another organism such as a fungus or another yeast, may be co-expressed in the yeast host cell as an auxiliary protein to assist in the folding of the protein of interest.
  • auxiliary proteins or chaparones may include a spliced version of Homologous to Atf/Crebl (HAC1; YFL031W), an ATPase such as KARyogamy 2 (KAR2; YJL034W), a glutathione peroxidase such as a phospholipid hydroperoxide glutathione peroxidase, for example glutathione peroxidase (GPX1; YKL026C), and proteins that may enhance translocation of the protein of interest out of the yeast host cell into the culture medium such as SECretory 1 (SEC1; YDR164C) and Suppressor of Loss of Yptl 1 (SLY1; YDR189W).
  • SECretory 1 SEC1; YDR164C
  • Suppressor of Loss of Yptl 1 SLY1; YDR189W
  • Tuning of expression is required when building synthetic pathways within organisms such as yeasts.
  • tuning of individual protein expression levels is desired to optimize many industrial biotechnological processes.
  • the promoter region may be changed to fine tune the transcription level and consequently the amount of protein produced. Fine tuning of individual expression levels of proteins that function in a cascade may enhance the overall yield of the final product.
  • Such pathways include, for example, the production of a biofuel such as ethanol, 1-butanol and isopropanol, for which enzymes such as acetyl-CoA C- acyltransferase (EC 2.3.1.16; for example Peroxisomal Oxoacyl Thiolase 1 (POTI; YIL160C), acetoacetyl-CoA transferase, for example ERGosterol biosynthesis 10 (ERG 10; YPL028W), and an acetoacetate decarboxylase play an important role (Nandy and Srivastava, 2018.
  • acetyl-CoA C- acyltransferase EC 2.3.1.16
  • POTI Peroxisomal Oxoacyl Thiolase 1
  • acetoacetyl-CoA transferase for example ERGosterol biosynthesis 10 (ERG 10; YPL028W)
  • an acetoacetate decarboxylase play an important role
  • Microbiol Res 207: 83-90 the breakdown of a carbohydrate such as cellulose, which includes the tuning of expression levels of several enzymes including, for example, endoglucanases and cellulase (Claes et al., 2020. Metabolic Engineering 59: 131-141), and production of a biopolyester such as polyhydroxyalkanoates (PHA), for which expression of, for example, B-ketothiolase, NADPH-linked acetoacetyl-CoA reductase and PHA synthase, needs to be carefully finetuned (Terentiev et al., 2004. Applied Microbiol Biotech 64: 376-381).
  • PHA polyhydroxyalkanoates
  • tocochromanol which term refers to amphipathic molecules with a hydrophobic isoprenoid- derived hydrocarbon tail and a polar aromatic head obtained from the shikimate pathway, and which include a, B, y, 5-tocopherols and tocotrienols.
  • Tocotrienols are formed from two precursors, homogentisic acid (HGA) and geranylgeranyl pyrophosphate (GGPP).
  • HGA is synthesized from 4-hydroxyphenylpyruvate (4-HPP) under catalysis of 4- hydroxyphenylpyruvate dioxygenase (HPPD), while GGPP is derived from 2C- Methyl-D-erythritol-4-phosphate (MEP) pathway.
  • HPPD 4-hydroxyphenylpyruvate dioxygenase
  • GGPP is derived from 2C- Methyl-D-erythritol-4-phosphate (MEP) pathway.
  • alkaloids have a wide range of pharmacological activities including antimalarial (e.g. quinine), antiasthma (e.g. ephedrine), anticancer (e.g. homoharringtonine), cholinomimetic (e.g. galantamine), vasodilatory (e.g. vincamine), anti arrhythmic (e.g. quinidine), analgesic (e.g. morphine), antibacterial (e.g. chelerythrine), and antihyper glycemic activities (e.g. piperine).
  • antimalarial e.g. quinine
  • antiasthma e.g. ephedrine
  • anticancer e.g. homoharringtonine
  • cholinomimetic e.g. galantamine
  • vasodilatory e.g. vincamine
  • anti arrhythmic e.g. quinidine
  • analgesic e.g. morphine
  • Example 1 Engineering of synthetic promoters by rational shuffling method Engineered promoter sequences were shuffled in the following way. One or two core sequences with a TATA-Box element or TATA- Boxdike element at position -90 ⁇ 15 bp and/or -160 ⁇ 15 bp were linked together without changing the relation of the core promoter to the start codon ATG. TATA-Box-like element were changed to TATA-Box sequences.
  • a first enhancer region was linked to the core element in a way that the binding sites of Saccharomyces cerevisiae transcription factor AZFI (5’-AAMRGMA) or MSN 4 (5’-RVCCCCYR) start a position -350 bp.
  • a second enhancer element exhibit GCRl-bs (5’-WGGAWGMY), GCR2-bs (5’- WGGAAGNM), or PHDl-bs (5’-VMTGCRKV) at position -600 bp, wherein -bs denotes binding sequence. Thereby the elements were not set at their natural distance to the start codon. Additional point mutations were set to partly prevent specific enzyme cleavage sites within the promoter sequences.
  • the different promoter sequences were ordered as synthetic strings or gene product from Twist Bioscience (San Francisco, CA, USA) or Invitrogen GeneArt Gene Synthesis (Thermo Fisher Scientific Inc.) with appropriate sequence overhang for the dedicated vector system.
  • the engineered promoter sequences and the reference promoter of Gen GAP1 were cloned via Xmal and EcoRI restriction sites into an expression vector (pKN95). This contains the yellow fluorescent protein YFP (Venus) gene in addition to a kanMX gene as a selection marker.
  • Expression vectors and empty vector (pKNl; Nord et al., 1997. Nature Biotech 15: 772-777) were transformed into competent K. phaffii cells by electroporation. To characterize the YFP expression under control of different promoters, all strains were cultivated as biological triplicates in deepwell plates.
  • the cultivation was performed at 28 °C and 70 % humidity in a volume of 1.5 ml YPD medium (1 % w/v yeast extract, 2 % w/v peptone and 2 % w/v glucose), supplemented with geneticin (G418; 500 pg/ml).
  • the medium was inoculated with three colonies of each respective strain. After 90 h, the samples were diluted 1:20 with fresh medium. 200 pl of each sample was pipetted into optical bottom plates (Thermo Scientific 96 well, black) in technical triplicates. Fluorescence (excitation 485 nm I emission 520 nm) and GD600 were measured in plate reader (FLUOstar Omega, BMG Labtech).
  • the carbon source was additional exchanged to provide glycerol and/or methanol instead of glucose.
  • the measured fluorescence was normalized to OD600 and the reference promoter GAP1 was set to 1.
  • the promoters EPK1 to 5 were designed at a later stage based on the good promoters. See Figure 1.
  • the promoters EPK18 to 23 showed a lower fluorescence intensity relative to reference promoter GAP1, so these are interesting for other applications like the expression of auxiliary enzymes.
  • individual genes can be up or down regulated.
  • Figure 3 shows the GFP fluorescence normalized to the GD600 after 24 h.
  • the reference promoter TEF1 was set at 1.
  • the strain with the construct EPK6 had a comparable fluorescence intensity than the TEF1 control.
  • the strains bearing the promoters EPK7, EPK8, EPK9 EPK10, EPK11 and EPK12 showed a significant stronger fluorescence than strains with reference promoter ( Figure 3A).
  • the strains bearing the promoters EPK27, EPK28, EPK29 EPK30, EPK31, EPK32 and EPK33 showed a similar fluorescence to strains with effective reference promoter (Figure 3B).
  • the different promoter fragments and the reference promoter PGK1 were cloned in front of the YFP gene into a 2g expression vector containing an uracil cassette as a selective marker (Lee et al. 2015. ACS Synth. Biol. 2015, 4, 9, 975-986).
  • These YFP expression vectors were chemically transformed into competent S. cerevisiae (CEN.PK2 1C) cells with the Li-Acetate, single stranded carrier DNA transformation protocol according to Gietz and Schiestl, 2007 (Gietz and Schiestl, 2007. Nat Protoc 2: 31-34).
  • CEN.PK2 1C competent S. cerevisiae
  • Example 5 Application of synthetic promoters for metabolic engineering of S. cerevisiae: tocochromanol production a. Concent for regulation of gene expression for tocochromanol production in yeast
  • the group of tocochromanols comprises tocotrienols and tocopherols, commonly known as Vitamin E, and are naturally produced by photo autotrophic organisms. Due to rising demands in food, feed and cosmetic industry a sustainable production process for tocochromanols is required. The goal of these experiments is to obtain microorganisms as biotechnological production hosts.
  • Tocochromanols consist of a chromanol group, which is derived from homogentisic acid, and an isoprene chain that is derived from geranylgeranyl-diphosphate (GGPP). These two precursors can be converted into all Vitamin E isoforms by heterologous genes from plants and cyanobacteria.
  • ARO3 UniProt #P14843
  • ARO4 P32449
  • these enzymes should be expressed in a feedback resistant version (Aro3K222L, in which a lysine (K) at position 222 is altered into a Leucine(L); and Aro4K229L, in which a lysine (K) at position 229 is altered into a Leucine (L)).
  • ARO1 which encodes a pentafunctional enzyme
  • ARO7 which encodes a pentafunctional enzyme
  • ARO7 P32178
  • TRP2 P00899
  • PHA2 P32452
  • genes of degrading pathways need to be deleted or downregulated (ARO8 (P53090), ARO9 (P38840), ARO10 (Q06408) and PDC5 (P16467)).
  • HPP hydroxyphenylpyruvate
  • the second precursor of the isoprene chain is geranylgeranyl- diphosphate (GGPP).
  • GGPP geranylgeranyl- diphosphate
  • HMG1 P12683
  • BTS1 Q 12051
  • Q1L6K3 a stronger heterologous variant like crtE from Xanthophyllomyces dendrorhous
  • the two precursors homogentisic acid and GGPP will be prenylated by a heterologous homogentisat phytyl-transferase (HPT) and further cyclized be the tocopherol cyclase (TC) for tocotrienol production.
  • HPT heterologous homogentisat phytyl-transferase
  • TC tocopherol cyclase
  • GGPP needs to be reduced by a heterologous GGPP reductase into phytyl-diphosphat (PDP).
  • y-TM7 y tocopherol methyltransferase
  • MPBQMT 2-methyl-6-phytylbenzoquinol methyltransferase
  • the synthetic promoter EPK14 was used in a project of metabolic engineering of S. cerevisiae for enhanced geranylgeraniol (GGOH) production.
  • Two different production strains JBY6 and JBY12 were generated from the modified CEN.PK2- 1C strain (Entian and Kbtter, 2007. Methods Microbiol 36: 629-666).
  • the enzyme geranyl- geranyl diphosphate synthase (GGPPS) which is encoded by BTS1 was shown to be crucial for GGOH production from GGPP.
  • the two strains JBY6 (leu2A::pPGKl-BTSl-tADHl) and JBY12 (hoA::EPK14-BTSl-tADHl) were created by integration of a BTS1 overexpression cassette with either the reference promoter PGK1 (JBY6) or the synthetic promoter EPK14 (JBY12).
  • the strains were verified on selective agar plates and by PCR methods. Since BTS1 is still limiting the GGOH production, the GGPPS was additionally overexpressed on 2q plasmids. All strains were cultivated for 144 h in 50 ml selective synthetic minimal medium + 2 % glucose in biological duplicates in shake flasks (30 °C, 180 rpm).
  • the strains JBY6, harbouring the PGK1 promoter and JBY12, bearing the synthetic promoter EPK14 were able to produce similar amounts of GGOH ( ⁇ 5 mg/L). Moreover, if BTS1 is additionally expressed on 2p plasmids the strain JBY12 is producing 18.55 mg/L whilst JBY6 did only produce 12.52 mg/L. This shows, exemplary for EPK14, that the synthetic promoters are applicable for gene expression in S. cerevisiae.
  • one of the weak promoters can be used to downregulate the competing ergosterol pathway. Either, farnesyl-diphosphate can be converted into the desired geranylgeranyl-diphosphate or into a precursor of ergosterol by ERG9 (P29704).
  • ERG9 P29704
  • the strong promoter EPK14 already leads to higher expression of BTS1, but the effect of upregulation can be enhanced by using one of the weak promoters so weaken the ERG9 expression and channel the flux from farnesyl-diphosphate into geranylgeranyl-diphosphate production.
  • the following example shows the applicability of the synthetic promoter EPK15 for heterologous gene expression in S. cerevisiae.
  • a yeast strain was constructed which is producing the two intermediates: homogentisic acid and GGPP.
  • two heterologous enzymes, the prenylase HPT (P73726)and the cyclase VTE1 (829413) are needed to form 5-tocotrienol. Both heterologous enzymes were integrated into the leu2 locus.
  • the synthetic promoter EPK15 was used in front of the heterologous HPT gene of Synechocystis spec, and a truncated version of the VTE1 (deletion of the first 47 amino acids) gene was expressed under the control of PGK1 (JBY20).
  • This strain was cultivated in biological triplicates in 50 ml YPD medium + 15 ml dodecane overlay at 30 °C and 180 rpm. After 144 h, 1 ml of the dodecane phase was harvested and measured in the HPLC Bio-LC (Dionex) using the Agilent Zorbax SB-C8 column (4.6 x 150 mm, 3.5 pm).
  • Buffer A (ddH2O + 0.1 % formic acid) and buffer B (acetonitrile + 0.1 % formic acid) were run in a gradient up to 100 % buffer B for 40 min and delta tocotrienol was measured using UV detection at 300 nm. Furthermore, 5- tocotrienol was identified by mass determination in the LC-MS ([M+H]+ 397.3101). Using this strain, delta-tocotrienol was produced by S. cerevisiae, which gives another example for the usage of synthetic promoters for metabolic engineering in yeast.
  • Example 6 Synthetic promoters for expression of App A from Escherichia coli in K. phaffii
  • the promoters EPK2 and EPK3, and the reference promoter GAP1 were cloned into an expression vector via the restriction sites Xmal and EcoRI.
  • This vector contains the AppA (P07102, amino acids 23-432) gene from E. coli in addition to a kanMX gene as a selection marker.
  • AppA expression vectors as well as the empty vector were transformed into electro-competent K. phaffii cells. After expression of the enzyme, it is secreted into the culture supernatant so that phytase activity can be measured in the medium. The activity of phytase can therefore be used as a direct signal of promoter strength.
  • the supernatant was diluted 1:80 with a sodium acetate buffer (250 mM HAc-NaAc buffer, pH 4.5 containing 0.01 % Tween20). Of this, 800 pl of phytate solution (7.5 mmol/1) was added to 400 pl. After incubation for 30 min at 37 °C, the reaction was stopped and mixed thoroughly. This was followed by incubation at room temperature (1 min) and then centrifugation of the reaction mixture at llOOOxg. The enzyme activity was measured in triplicates in 96-well plates. The absorbance were measured at 415 nm in a plate reader (FLUOstar Omega, BMG Labtech).
  • the measured activity was normalized to OD600 and the reference promoter GAP1 was set to 1 ( Figure 7).
  • the carbon source was exchanged to provide glycerol or methanol instead of glucose.
  • promoters are very strong. Some of the promoters showed weaker fluorescence intensities compared to the reference promoter GAP1. These promoters are interesting for metabolic engineering, as being able to regulate multiple genes individually.
  • auxiliary enzymes such as PDI (B3VSN1). It is known that the enzyme PDI can increase the correct folding rate of recombinantly expressed proteins, especially in the presence of additional disulfide bridges.
  • auxiliary enzymes like ERV2 (C4R490), EUG1 (P32474), ERO1 (A0A1B2J869) and MPDI (C4QVB2).
  • the invertase SUC2 (P00724) is an enzyme with which sucrose would be available as a carbon sources.
  • AAAAC AT C AAGAAC AAAC AAGC T C AACT T GTCTTTTC T AAG AAC AAAGAAT AAAC AC AA AAAC AAAAAGT T T T T T T AAT T T T AAT C AAAAA

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Abstract

L'invention concerne une région promotrice artificielle de levure, comprenant une boîte TATA à la position -90 ± 15 nucléotides (nt) et/ou une boîte TATA à la position -160 ± 15 nt, un élément activateur à la position -350 ± 25 nt, et un second élément activateur à la position -600 ± 50 nt. L'invention concerne également une construction d'expression comprenant la région promotrice artificielle de levure, une cellule hôte de levure, comprenant la région promotrice artificielle de levure, et des procédés de production d'une protéine d'intérêt dans une cellule hôte de levure en utilisant la région promotrice artificielle de levure.
PCT/EP2022/082939 2021-11-23 2022-11-23 Régions promotrices artificielles de levure WO2023094429A1 (fr)

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US20180371468A1 (en) * 2016-02-22 2018-12-27 Teknologian Tutkimuskeskus Vtt Oy Expression system for eukaryotic organisms
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